Shandong Provincial Key Laboratory of Energy Genetics, Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology , Chinese Academy of Sciences , 266101 Qingdao , People's Republic of China.
State Key Laboratory of Microbial Technology , Shandong University , Jinan , 250100 , People's Republic of China.
Biomacromolecules. 2018 May 14;19(5):1686-1696. doi: 10.1021/acs.biomac.8b00340. Epub 2018 Apr 11.
Processive hydrolysis of crystalline cellulose by cellulases is a critical step for lignocellulose deconstruction. The classic Trichoderma reesei exoglucanase TrCel7A, which has a closed active-site tunnel, starts each processive run by threading the tunnel with a cellulose chain. Loop regions are necessary for tunnel conformation, resulting in weak thermostability of fungal exoglucanases. However, endoglucanase CcCel9A, from the thermophilic bacterium Clostridium cellulosi, comprises a glycoside hydrolase (GH) family 9 module with an open cleft and five carbohydrate-binding modules (CBMs) and hydrolyzes crystalline cellulose processively. How CcCel9A and other similar GH9 enzymes bind to the smooth surface of crystalline cellulose to achieve processivity is still unknown. Our results demonstrate that the C-terminal CBM3b and three CBMX2s enhance productive adsorption to cellulose, while the CBM3c adjacent to the GH9 is tightly bound to 11 glucosyl units, thereby extending the catalytic cleft to 17 subsites, which facilitates decrystallization by forming a supramodular binding surface. In the open cleft, the strong interaction forces between substrate-binding subsites and glucosyl rings enable cleavage of the hydrogen bonds and extraction of a single cellulose chain. In addition, subsite -4 is capable of drawing the chain to its favored location. Cellotetraose is released from the open cleft as the initial product to achieve high processivity, which is further hydrolyzed to cellotriose, cellobiose and glucose by the catalytic cleft of the endoglucanase. On this basis, we propose a wirewalking mode for processive degradation of crystalline cellulose by an endoglucanase, which provides insights for rational design of industrial cellulases.
纤维素酶对结晶纤维素的连续水解是木质纤维素解构的关键步骤。经典的里氏木霉外切葡聚糖酶 TrCel7A 具有封闭的活性位点隧道，通过将纤维素链穿过隧道开始每个连续的作用。环区对于隧道构象是必需的，导致真菌外切葡聚糖酶的热稳定性较弱。然而，来自嗜热细菌纤维素梭菌的内切葡聚糖酶 CcCel9A 包含一个糖苷水解酶 (GH) 家族 9 模块，具有开放的裂缝和五个碳水化合物结合模块 (CBM)，并连续水解结晶纤维素。CcCel9A 和其他类似的 GH9 酶如何结合到结晶纤维素的光滑表面上以实现连续性仍然未知。我们的结果表明，C 端 CBM3b 和三个 CBMX2 增强了对纤维素的有效吸附，而与 GH9 相邻的 CBM3c 紧密结合到 11 个葡萄糖基单元上，从而将催化裂缝扩展到 17 个亚位点，这有助于通过形成超模块结合表面进行去结晶。在开放的裂缝中，底物结合亚位点和葡萄糖基环之间的强相互作用力使氢键断裂，并提取出单个纤维素链。此外，亚位点 -4 能够将链拉到其首选位置。纤维四糖从开放的裂缝中作为初始产物释放出来，以实现高连续性，然后通过内切葡聚糖酶的催化裂缝进一步水解为纤维三糖、纤维二糖和葡萄糖。在此基础上，我们提出了一种内切葡聚糖酶对结晶纤维素进行连续降解的“走丝”模式，为工业纤维素酶的合理设计提供了思路。
